Polymer microfilters, devices comprising the same, methods of manufacturing the same, and uses thereof

ABSTRACT

A microfilter having a hydrophilic surface and suited for size-based capture and analysis of cells, such as circulating cancer cells, from whole blood and other human fluids is disclosed. The filter material is photo-definable, allowing the formation of precision pores by UV lithography. Exemplary embodiments provide a device that combines a microfilter with 3D nanotopography in culture scaffolds that mimic the 3D in vivo environment to better facilitate growth of captured cells.

This application is a continuation in part of, and claims priority to,the following prior applications the contents of all of which are herebyincorporated by reference herein: “POLYMER MICROFILTERS, DEVICESCOMPRISING THE SAME, METHODS OF MANUFACTURING THE SAME, AND USESTHEREOF”, U.S. patent application Ser. No. 14/213,183, filed on Mar. 14,2014 claiming priority to U.S. Provisional Patent Application No.61/794,628, filed Mar. 15, 2013; “POLYMER MICROFILTERS AND METHODS OFMANUFACTURING THE SAME”, U.S. patent application Ser. No. 13/696,139,filed on Nov. 5, 2012, which is a 371 of PCT/US11/30966, filed Apr. 1,2011 claiming priority to two U.S. Provisional Patent Applications No.61/330,819, filed May 3, 2010 and No. 61/377,797, filed Aug. 27, 2010;and “POLYMER MICROFILTRATION DEVICES, METHODS OF MANUFACTURING THE SAMEAND THE USES OF THE MICROFILTRATION DEVICES”, U.S. patent applicationSer. No. 16/400,600, filed May 6, 2019, which is a continuation of U.S.patent application Ser. No. 13/854,003 filed on Mar. 29, 2013, which isa continuation in part of PCT/US12/66390 filed on Nov. 21, 2012,claiming priority to three U.S. Provisional Patent Applications No.61/562,404 filed Nov. 21, 2011, No. 61/618,641 filed Mar. 30, 2012, andNo. 61/654,636 filed Jun. 1, 2012.

BACKGROUND

Circulating tumor cells (CTCs) disseminated into peripheral blood from aprimary or metastatic tumor can be used to phenotype and determine anorgan of disease for diagnosis, to perform mutational studies to choosea targeted therapy, to monitor therapy effectiveness, to detectrecurrence of disease, and to provide prognostic survival information ofsolid malignancies. Due to this wide variety of potential applications,a large number of techniques have been developed to enrich for CTCs.Enrichment/capture of CTCs is challenging, because of their extremerarity, as few as 1 in 7.5 mL of blood containing 10⁹ blood cells. Sincetumor cells are generally larger than blood cells, filtration of CTCshas been considered as long ago as 1964 by S. H. Seal of Memorial SloanKettering Cancer Center. In the past 15 years, filtration of CTCs hasmade significant advances. Even though it has been shown that filtrationtechniques are the most rapid and straightforward method to captureCTCs, filter choices were limited and less than ideal. At the present,track-etch polycarbonate filters are the only products commerciallyavailable for CTC applications. Track etch filters are used in productsby ScreenCell® and Rarecells SAS. Since the pores in track-etch filtersare distributed randomly, pores can overlap, resulting in variable poresize and low capture efficiency. Each track-etch filter is somewhatdifferent from the others, so the standard deviation of capture is high.In an effort to minimize this pore overlap, porosity is typically keptlow (3-5%), resulting in slow filtration and high nonspecific cellcontamination on the filter.

Lithographic fabrication methods are able to produce uniform andprecisely-patterned microfilters for CTC capture. This method has beenaccomplished in various academic settings using parylene, silicon,silicon nitride and nickel as the filter material. In each case,photolithographic membranes showed good clinical applicability whentested for CTC capture from patient blood samples. Most notable isparylene microfilters showing high CTC capture, which compared favorablyagainst the classic CellSearch® CTC test (Veridex). Parylene material,however, is auto-fluorescent, and the parylene microfilters do not lieflat on microscope slides, complicating microscope imaging. Furthermore,the parylene filter fabrication method is a multi-step process,rendering it unsuitable for cost-effective volume production. Thealternative membrane materials, including silicon, silicon nitride andnickel, are not transparent, the fabrication methods are hindered byhigh cost and limited scalability, which has prevented widespreadtesting, and clinical implementation is complicated. Further, as many ofthese materials are fragile or difficult to handle, support structuresare needed to stabilize the membrane during filtration and analysis.

Given the limitations of existing filters, there is a need to developnew types of filters with improved characteristics. The presentinvention is directed to this and other important goals.

For some application, it is desirable for the microfilters to havesurface treatment to (1) improving methods to attach antibodies,ligands, proteins, DNA, etc to the surface and (2) to producenanosurface features. Some applications of nanosurface modifications are(a) changes the surface to be hydrophilic and (b) to enable 3D culture.

It is now understood and accepted that 2D culture induces cellularcharacteristics that differ significantly from those of tumors growingin vivo. It was shown that cell culture plates with 3D nanoimprintedscaffolds provide reproducible and significantly improved cell cultureby facilitating cellular migration, intercellular adhesion, cellularviability, and proliferation, thus replicating the key features oftumors developing in vivo.

BRIEF SUMMARY

The present inventions is directed to microfilters having a hydrophilicsurface that can be used to collect selected components, such as cells,from a fluid, such as a bodily fluid, including whole blood, urine, bonemarrow, bladder wash, rectal brushings, fecal matter, saliva, cordblood, spinal and cerebral fluids, and other body fluids. The presentinventions is also directed to the methods of using the microfilters inthe removal and/or collection of materials, such as cells, from a fluid.The present invention is further directed devices comprising themicrofilters, and to the methods of manufacturing the microfilters.

In a first embodiment of the present invention, a microfilter having ahydrophilic surface and suited for size based capture and analysis ofcells, such as CTCs, from whole blood and other human fluids isprovided. The filter material is photo-definable, allowing the formationof precision pores by UV lithography. The filter material is alsosubject to modification that results in at least one surface of themicrofilter being hydrophilic. In one aspect of this embodiment, theinvention is directed to a microfilter comprising a polymer layer formedfrom a photo-definable dry film, wherein a surface of the polymer layeris modified to be hydrophilic, and a plurality of apertures eachextending through the polymer layer. In aspects of this embodiment,wherein the film is an epoxy-based photo-definable dry film. In aspectsof this embodiment, the modification raises the surface energy of thepolymer layer or produces a rough nanosurface on the polymer layer.

In further aspects of the first embodiment, the microfilter displays atleast one analyte capture element on a surface of the polymer layer. Theanalyte capture element may comprise one or more of a polypeptide,nucleic acid, carbohydrate, and lipid. As a specific, non-limitingexample, the analyte capture element may comprise an antibody withbinding specificity for one or more of (i) EpCAM, (ii) MUC-1, (iii) bothEpCAM and MUC-1, (iv) CD24, (v) CD34, (vi) CD44, (vii) CD133, and (viii)CD166.

In a second embodiment of the invention, a device that comprises amicrofilter of the invention in a scaffold for use in tissue culture isprovided. The device allows the 3D in vivo environment to be mimicked invitro, thus better facilitating growth of captured cells. In aspects ofthis embodiment, such devices can facilitate a rapid, gentle, easy workflow to culture CTCs.

In a third embodiment of the invention, the following are provided: (a)methods to produce nanosurface structures on polymer sheets and films,and on polymer microfilters, that impart a hydrophilic characteristic toa surface of the sheet, film or microfilter, (b) applications to usenanosurface polymer materials for culturing cells, (c) culture platesand devices using the nanosurface sheets, films or microfilters forculture of cells, (d) applications of cell capture from body fluids withstandard and nanosurface structured microfilters, and (e) coating ofanalyte capture elements on microfilters, and (f) applications ofmicrofilters coated with analyte capture elements.

In a fourth embodiment, the present invention is directed to methods ofusing the microfilters of the invention in the collection of selectedcomponents from a fluid, such as a biological fluid. For example, in oneaspect of this embodiment the invention is directed to a method of usinga microfilter by passing a fluid through a plurality of apertures of amicrofilter formed from an photo-definable dry film, wherein themicrofilter has sufficient strength and flexibility to filter the fluid,and wherein the apertures are sized to allow passage of a first type ofcomponent in the fluid and to substantially prevent passage of a secondtype of component in the fluid. In a related aspect, the method furthercomprises collecting the second type of component in the fluid from thefilter and performing one or more of identification, immunofluorescence,enumeration, sequencing, PCR, fluorescence in situ hybridization, mRNAin situ hybridization, other molecular characterizations,immunohistochemistry, histopathological staining, flow cytometry, imageanalysis, enzymatic assays, gene expression profiling analysis,erythrocyte deformability, white blood cell reactions, efficacy tests oftherapeutics, culturing of enriched cells, and therapeutic use ofenriched rare cells on the collected second component.

Fluids that might be used in conjunction with the methods include, butare not limited to, blood, urine, bone marrow, bladder wash, rectalbrushings, fecal matter, saliva, cord blood, spinal and cerebral fluids,and other body fluids.

The second type of component in the fluid includes, but is not limitedto, at least one member selected from the group consisting of:circulating tumor cells, tumor cells, epithelial-mesenchymal transitioncells, CAMLs, white blood cells, B-cells, T-cells, circulating fetalcells in mother's blood, circulating endothelial cells, stromal cells,mesenchymal cells, endothelial cells, epithelial cells, stem cells,hematopoietic and non-hematopoietic cells, analytes bound to latex beadsor an antigen-induced particle agglutination.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the present invention and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following detaileddescription when considered in connection with the accompanyingdrawings, wherein:

FIG. 1 is scanning electron micrograph (SEM) showing an example ofmicrofilter fabricated based on the method and material described in thecross reference patents.

FIGS. 2A-2D show examples of nanoscale features on polymer surfacesetched by ME.

FIG. 3 shows an example of a microfilter after ME showing a pore andnanosurface topography.

FIG. 4 shows an example of a microfilter after energetic neutral oxygenatom etching showing pores and nanosurface topography.

FIG. 5A shows an example of an anodic aluminum oxide (AAO) templateformed above a polymer substrate. AAO has nanopores.

FIG. 5B shows an example of polymer surface after RIE through AAO.

FIG. 6 shows an example of nanoscale surface topography microfilter withpores produced by imprinting using rough metal surface as the mold.

FIG. 7 shows an example of lithographically produced microwells on topof a microfilter.

FIG. 8A shows an example of T24 cell culture on chamber slide showingDAPI nucleus in white on black background.

FIG. 8B shows the same T24 cell culture on chamber slide showing amerged color image of DAPI nucleus in blue and cytokeratin (CK) 8 and 18in green. The CK expression is very weak.

FIG. 9A shows an example of T24 cell culture on RIE treatedphoto-definable dry film showing DAPI nucleus in white on blackbackground in the form of a cluster.

FIG. 9B shows the same T24 cell culture on RIE treated photo-definabledry film showing a merged color image of DAPI nucleus in blue and CK 8and 18 in green. The CK expression is high.

DETAILED DESCRIPTION

Referring now to the drawings, wherein like reference numerals designateidentical or corresponding parts throughout the several views, exemplaryaspects of the present invention are shown in schematic detail.

The matters defined in the description such as a detailed constructionand elements are nothing but the ones provided to assist in acomprehensive understanding of the invention. Accordingly, those ofordinary skill in the art will recognize that various changes andmodifications of the exemplary aspects described herein can be madewithout departing from the scope and spirit of the invention. Also,well-known functions or constructions are omitted for clarity andconciseness. Some exemplary aspects of the present invention aredescribed below in the context of commercial applications. Suchexemplary implementations are not intended to limit the scope of thepresent invention, which is defined in the appended claims

Aspects of the present invention are generally directed to a microfiltercomprising a polymer layer formed from a photo-definable dry film, suchas an epoxy-based photo-definable dry film. The microfilter includes aplurality of apertures each extending through the polymer layer.Further, the polymer layer is modified to be hydrophilic. In certainexemplary aspects, the microfilter may be formed by exposing the dryfilm to energy through a mask and developing the exposed dry film. Insome exemplary aspects, the dry film may be exposed to energy in theform of ultraviolet (UV) light. In other exemplary aspects, the dry filmmay be exposed to energy in the form of X-rays. In certain exemplaryaspects, the polymer layer has sufficient strength and flexibility tofilter liquid. In some exemplary aspects, the apertures are sized toallow passage of a first type of bodily fluid cell and to preventpassage of a second type of bodily fluid cell.

According to exemplary implementations of the present disclosure, amicrofilter can comprise a polymer layer formed from an epoxy-basednegative photo-definable dry film. According to further exemplaryimplementations of the present disclosure, the apertures of themicrofilter can be essentially of any shape or configuration such asround, oval, racetrack, or rectangle, or any combination thereof. In yetother exemplary aspects, the polymer layer of the microfilter can have auniform thickness of 5 to 100 microns. In still further exemplaryaspects, the polymer layer of the microfilter can have a uniformthickness of 10 μm. In yet further exemplary aspects, the apertures canround with a diameter of 5-20 μm. Any combinations of such polymerlayer, aperture features and configurations are within the scope for amicrofilter structure of the present disclosure.

Specifically, in certain exemplary aspects, the microfilter may be usedto perform assays on bodily fluids. In some exemplary aspects, themicrofilter may be used to isolate and detect large rare cells from abodily fluid. In certain exemplary aspects, the microfilter may be usedto collect circulating tumor cells (CTCs) from peripheral blood fromcancer patients passed through the microfilter. In certain exemplaryaspects, the microfilter may be used to collect circulating endothelialcells, fetal cells and other large cells from the blood and body fluids.In certain exemplary aspects, the microfilter may be used to collectlarge cells from processed tissue samples, such as bone marrows. In someexemplary aspects, cells collected using the microfilter may be used indownstream processes such as cell identification, enumeration,characterization, culturing, etc.

More specifically, in certain exemplary aspects, multiple layers ofphoto-definable dry film, such as an epoxy-based photo-definable dryfilm, may be exposed to energy simultaneously for scaled production ofmicrofilters. In some exemplary aspects, a stack of photo-definable dryfilm layers is provided, and all of the dry film layers in the stack areexposed to energy simultaneously. In some exemplary aspects, a dry filmstructure including photo-definable dry film disposed on a substrate isprovided in the form of a roll. In such exemplary aspects, a portion ofthe structure may be unrolled for exposure of the dry film to energy. Incertain exemplary aspects, portions of a plurality of rolls may beexposed to energy simultaneously.

FIG. 1 is scanning electron micrograph (SEM) of microfilter fabricatedbased on the known techniques. The surface is smooth, shiny andhydrophobic. The contact angle is approximately 90 degrees. Thehydrophobic property of the material allows performing assays withreagents staying above the filter without the reagents leaking through.However, the hydrophobic nature of the filter is also problematic whenit is desired to have a filter through which fluids easily pass, e.g., amicrofilter with hydrophilic surface characteristics. For someapplications, it is desirable to modify a surface of the microfilter tohave hydrophilic characteristics via, for example, increasing thesurface energy of a surface of the microfilter and/or altering thesurface topography of a surface of the microfilter through variousmethods of surface treatment.

Surface Modification Methods and Resultant Microfilters

The surface of a microfilter may be modified to impart a hydrophiliccharacteristic through methods of surface treatment. The most commonmethods of surface treatment are based on a principle of high voltagedischarge in air without changing the topography of the surface. Whenthe microfilter is placed in the discharge path, the electrons generatedin the discharge impact the surface creating reactive free radicals.These free radicals in the presence of oxygen can react rapidly to formvarious chemical function groups on the microfilter surface. This raisesthe surface energy of the microfilter. It changes the microfilter fromhydrophobic to hydrophilic. Surface treatment can improve wettability ofthe microfilter by raising the material's surface energy and positivelyaffect adhesive characteristics by creating bonding sites. An example ofhigh voltage discharge is corona discharge.

Some of the applications of microfilters treated by corona dischargeare: (i) flow of fluid through small pores with less resistance, (ii)cell morphologies may be better preserved when the use of small poresare required, (iii) better conjugation of analyte capture elements tothe microfilter, (iv) attachment of various surface modificationmaterials, and others.

Four additional methods of surface treatment are provided herein thatproduce surface modifications on polymer microfilters and that serve toincrease the hydrophilicity of the surface: (a) reactive ion etching,(b) energetic neutral oxygen atoms etching, (c) reactive ion etchingthrough anodic aluminum oxide (AAO) template, and (d) surfaceimprinting. These methods make a surface of a polymer layer rougher intexture. The 3D surface features produced by each method are differentbut they share the characteristic that the surface of the polymer layerthat has undergone treatment is rougher in texture than the surfaceprior to treatment. As with microfilters using polymer layers withsurfaces treated using corona discharge, microfilters using polymerlayers with surfaces treated to alter the 3D surface features alsoexhibit (i) increased flow of fluid through small pores with lessresistance, (ii) better preservation of cell morphologies, (iii) betterconjugation of analyte capture elements to the microfilter, and (iv)improved attachment of various surface modification materials.

Reactive Ion Etching (RIE) Method.

RIE utilizes chemically reactive plasma (high-energy ions) to removematerial from the surface of a polymer layer. This results in thecreation of a rough nanosurface on the polymer layer. Variations in theresulting etching of the surface are achieved depending on the materialto be etched and on the settings of RIE parameters. FIGS. 2A-D arescanning electron micrographs (SEMS) of examples of surfacemodifications produced by RIE on photo-definable dry-film without pores.FIG. 2D shows nanostructures with two different length scales.

RIE can be applied to microfilters, such as track etch microfilters,parylene microfilters, microfilters produced from photo-definable dryfilms, any filters made by polymer material as well as made from siliconwafers. FIG. 3 shows a SEM of a microfilter fabricated based on themethod and material described in the cross reference patents, followedby treatment by RIE showing nanosurface topography and a pore.

The surface treated by RIE becomes hydrophilic. The contact angle isalmost zero.

Energetic Neutral Oxygen Atom Etching.

Another method to produce a rough nanosurface on a polymer layer is toapply energetic neutral oxygen atom etching on the polymer surface withor without pores. To create a rough nanosurface on microfilters,energetic neutral oxygen atom etching is performed after themicrofilters are already formed but still attached to substrate. FIG. 4shows SEM of a microfilter treated by energetic neutral oxygen atomsshowing nanosurface topography and pores.

RIE Through a Nanoporous AAO Mask.

Another method to produce a rough nanosurface on a polymer layer using aporous metal material as a mask. (i) One example of a mask is to utilizeAAO. AAO template is fabricated on the resist surface by deposition andanodizing of −1 μm-thick Al film according to recipe. FIG. 5A is a SEMof the AAO template above the surface of the polymer material. Surfacerelief is obtained by RIE via AAO template followed by AAO removal inphosphoric acid solution. SEM of the resultant nanosurface structure isshown in FIG. 5B. (ii) Another group of porous materials for RIE aremicro magnetic beads and glass beads.

Nanoimprinting.

Another method to produce a rough nanosurface on a polymer layer is byimprinting the dry film on nanostructured surface. Using photo-definabledry films for microfilters, the substrate with rough nanosurface can beused. FIG. 6 is an SEM of microfilter produced by imprinting the dryfilm on the rough metal substrate. The nanosurface features is directlydependent on the mold.

For some applications, it is desirable to have wells formed above themicrofilters. For example of culture of cells in their individual well.A method to form the wells consists of laminated photo-definable dryfilms on surface of filter material with pores already formed.Microfilter-culture wells are fabricated using UV lithography, followedby development. After a hard bake, the microfilter device with wells canbe released from substrate. FIG. 7 shows an SEM of a microfilter withsquare wells.

3D Culture

Cell culture properties are highly dependent on the type of cell. It hasbeen shown that some cells growing in culture in clumps (3D) expressdifferent markers than the same cell line grown in a flat layer (2D) onthe culture plate. There has been a lot of research on findingconditions for 3D culture. A bladder cancer cell line, T24, was selectedto illustrate the effect of 2D and 3D culture.

When the microfilters or polymer materials of the present invention werecoated with fetal bovine serum (FBS) and bovine serum albumin (BSA), thebladder cancer cell line T24 grew similar to culturing on the standardculture chamber slide. However, if FBS or BSA can be eliminated, theculture process can be simplified.

When the polymer materials or microfilters of the present invention areuncoated, the T24 culture results become very different. FIG. 8A showsthe microscope imaging of the nuclei stained by DAPI of T24 cells grownon chamber slide. The cells grew flat in 2D format. The cells are imagedafter permeabilized and stained by cytokeratin (CK) 8 and 18 conjugatedto FITC dye. FIG. 8B shows the microscope imaging combining DAPI (blue)and CK 8, 18 (green). The cells show very low or no CK 8 and 18.

When T24 cells were cultured on photo-definable dry film polymer nottreated by RIE, T24 cells grew in 2D format similar to the results ofchamber slide.

In contrast, T24 cells grew in 3D clumps on photo-definable dry filmpolymer treated by low dose RIE. FIG. 9A shows the microscope imaging ofthe clump of nuclei stained by DAPI of T24 cells grown on ME treatedfilms. The cells are permeabilized and stained by cytokeratins (CK) 8and 18 conjugated to FITC. FIG. 9B shows the microscope imagingcombining DAPI (blue) and CK 8, 18 (green). The cells show very strongCK 8, 18.

It was also found that when cells were spiked into PBS followed byfiltration using RIE treated microfilter, FIG. 3, the cells grew in a 3Dformat.

In summary, it has been shown that the photo-definable dry film polymertreated with ME enabled 3D culture, and that the 3D cultured cellsbehaved differently than 2D cultured cells.

Culture Plates and Devices

Devices to implement 3D culture on chamber slides, and 6, 12, 24, 96 and384 well culture plates were prepared. Some variations of implementationare possible.

-   -   Place RIE etched polymers on the bottom of these wells. This        includes RIE etched photo-definable dry film polymer.    -   Place RIE etched polymers on the bottom of these wells coated        with FBS or BSA. This includes RIE etched photo-definable dry        film polymer.    -   Place RIE etched microfilters on the bottom of these well. This        includes RIE etched photo-definable dry film microfilter.    -   Place RIE etched microfilters on the bottom of these well coated        with FBS or BSA. This includes RIE etched photo-definable dry        film microfilter. This includes RIE etched photo-definable dry        film microfilter.    -   Place fibroblast cells, fibroblast cell fragments, other cells,        other cell fragments, or other culture reagents on the bottom of        the culture wells. Place RIE etched microfilters above that.    -   Cells can be captured on the RIE etched microfilter before        placing into culture plates.    -   Cells can be captured on FBS coated microfilters before placing        into culture plates.        Coating of Smooth Microfilters and Nanosurface Microfilters with        Analyte Capture Elements

As used herein, the term “analyte” is intended to mean a biologicalparticle. Biological particles include, for example, cells, tissues, ororganisms as well as fragments or components thereof. Specific examplesof biological particles include bacteria, spores, oocysts, cells,viruses, bacteriophage, membranes, nuclei, golgi, ribosomes,polypeptides, nucleic acid and other macromolecules. “Analyte complex”is intended to mean a biological particle or a group of biologicalparticles connected to analyte capture coating and/or other components,such as proteins, DNA, polymers, optical emission detection reagent,etc.

“Analyte capture” coating or elements are useful for selectivelyattaching or capturing a target analyte to microfilter. Attachment orcapture includes both solid or solution phase binding of an analyte toan analyte capture element. An analyte is attached or captured through asolid phase configuration when the analyte capture coating or element isimmobilized to a microfilter when contacted with an analyte. An analyteis attached or captured through a solution phase configuration when theanalyte capture coating or element is in solution when contacted with ananalyte. Subsequent immobilization of a bound analyte-analyte capturecoating or element complex to a microfilter completes attachment orcapture to the microfilter. In either configuration, either direct orindirect immobilization of the analyte capture coating or element to amicrofilter can occur. Direct immobilization refers to attachment of theanalyte capture coating or element to a microfilter allowing for captureof an analyte from solution to a solid phase. Immobilization of theanalyte capture coating or element can be directly to a microfiltersurface or through secondary binding partners such as linkers oraffinity reagents such as an antibody. Indirect binding refers toimmobilization of the analyte capture coating or element to amicrofilter. Analyte capture elements can form an analyte capturecomplex and become attached to the analyte capture surface on themicrofilter.

Moieties useful as an analyte capture coating or element in theinvention include biochemical, organic chemical or inorganic chemicalmolecular species and can be derived by natural, synthetic orrecombinant methods. Such moieties include, for example, macromoleculessuch as polypeptides, nucleic acids, carbohydrate and lipid. Specificexamples of polypeptides that can be used as an analyte capture coatingor element include, for example, an antibody, an antigen target for anantibody analyte, receptor, including a cell receptor, binding protein,a ligand or other affinity reagent to the target analyte. Specificexamples of nucleic acids that can be used as an analyte capture coatingor element include, for example, DNA, cDNA, or RNA of any length thatallow sufficient binding specificity. Accordingly, both polynucleotidesand oligonucleotides can be employed as an analyte capture coating orelement of the invention. Other specific examples of an analyte capturecoating or element include, for example, ganglioside, aptamer, ribozyme,enzyme, or antibiotic or other chemical compound. Analyte capturecoatings or elements can also include, for example, biological particlessuch as a cell, cell fragment, virus, bacteriophage or tissue. Analytecapture coatings or elements can additionally include, for example,chemical linkers or other chemical moieties that can be attached to amicrofilter and which exhibit selective binding activity toward a targetanalyte. Attachment to a microfilter can be performed by, for example,covalent or non-covalent interactions and can be reversible oressentially irreversible. Those moieties useful as an analyte capturecoating or element can similarly be employed as an secondary bindingpartner so long as the secondary binding partner recognizes the analytecapture coating or element rather than the target analyte. Specificexamples of an affinity binding reagent useful as a secondary bindingpartner is avidin, or streptavidin, or protein A where the analytecapture coating or element is conjugated with biotin or is an antibody,respectively. Similarly, selective binding of an analyte capturecoatings or element to a target analyte also can be performed by, forexample, covalent or non-covalent interactions. Specific examples of abiochemical analyte capture coating or element is an antibody. Aspecific example of a chemical analyte capture coating or element is aphotoactivatable linker. Other analyte capture coatings or elements thatcan be attached to a microfilter and which exhibit selective binding toa target analyte are known in the art and can be employed in the device,apparatus or methods of the invention given the teachings and guidanceprovided herein.

One exemplary form of microfilters manufactured in accordance withexemplary aspects of the present invention (i) standard microfilters and(ii) nanosurface topography microfilters are coated with analyte captureelements.

One specific exemplary form of the microfilters are microfilters coatedwith antibodies against EpCAM, MUC-1, and other surface markers are tocapture tumor cells from body fluids, such as blood, urine, bone marrow,bladder wash, rectal brushings, fecal matter, saliva, spinal andcerebral fluids, and other body fluids.

Another specific exemplary form of the microfilters coated withantibodies against CD24, CD44, CD133, CD166, and/or other surfacemarkers are to capture epithelial-mesenchymal transition (EMT) cellsfrom body fluids, such as blood, urine, bone marrow, bladder wash,rectal brushings, fecal matter, saliva, cord blood, spinal and cerebralfluids, and other body fluids.

Another specific exemplary form of the microfilters coated withantibodies against CD34, and/or other surface markers are to capturestem cells from body fluids, such as peripheral blood and cord blood.

Filtration Applications of Smooth Microfilters and NanosurfaceMicrofilters

Exemplary applications of the various forms of microfilters manufacturedin accordance with exemplary aspects of the present invention (e.g. (i)standard microfilters, (ii) nanosurface topography microfilters, (iii)standard microfilters coated with analyte capture elements, and (iv)nanosurface microfilters coated with analyte capture elements) are forprocessing body fluids, such as blood, urine, bone marrow, bladder wash,rectal brushings, fecal matter, saliva, spinal and cerebral fluids, andother body fluids. The analyte of interests in the body fluids arecirculating tumor cells, tumor cells, epithelial-mesenchymal transition(EMT) cells, CAMLs, white blood cells, B-cells, T-cells, circulatingfetal cells in mother's blood, circulating endothelial cells, stromalcells, mesenchymal cells, endothelial cells, epithelial cells, stemcells, hematopoietic and non-hematopoietic cells, analytes bound tolatex beads or an antigen-induced particle agglutination.

Another exemplary application of the microfilters manufactured inaccordance with exemplary aspects of the present invention (e.g. (i)standard microfilters, (ii) nanosurface topography microfilters, (iii)standard microfilters coated with analyte capture elements, and (iv)nanosurface microfilters coated with analyte capture elements) iscapturing circulating cancer associated macrophage-like cells (CAMLs)from peripheral blood. CAMLs have the following characteristics:

-   -   CAMLs have a large atypical nucleus; multiple individual nuclei        can be found in CAMLs, though enlarged fused nucleoli        approximately 14 μm to approximately 65 μm are common.    -   CAMLs may express at least CK 8, 18 or 19, and the CK is        diffused, or associated with vacuoles and/or ingested material.        CAMLs express markers associated with the type of cancer. Those        markers and CK are nearly uniform throughout the whole cell.    -   CAMLs are most of the time CD45 positive.    -   CAMLs are large, approximately 20 micron to approximately 300        micron in size.    -   CAMLs come in five distinct morphological shapes (spindle,        tadpole, round, oblong, or amorphous).    -   If CAML express EpCAM, EpCAM is diffused, or associated with        vacuoles and/or ingested material, and nearly uniform throughout        the whole cell, but not all CAML express EpCAM, because some        tumors express very low or no EpCAM.    -   CAML express markers associated with the markers of the tumor        origin; e.g., if the tumor is of prostate cancer origin and        expresses PSMA, then CAML from this patient also expresses PSMA.        Another example, if the primary tumor is of pancreatic origin        and expresses PDX-1, then CAML from this patient also expresses        PDX-1.    -   CAMLs express monocytic markers (e.g. CD11c, CD14) and        endothelial markers (e.g. CD146, CD202b, CD31). CAMLs also have        the ability to bind Fc fragments.

Another exemplary application of a microfilter manufactured inaccordance with exemplary aspects of the present invention (e.g. (i)standard microfilters, (ii) nanosurface topography microfilters, (iii)standard microfilters coated with analyte capture elements, and (iv)nanosurface microfilters coated with analyte capture elements) iscapturing circulating fetal cells in a mother's blood during weeks 11-12weeks of pregnancy. Such fetal cells may include primitive fetalnucleated red blood cells. Fetal cells circulating in the peripheralblood of pregnant women are a potential target for noninvasive geneticanalyses. They include epithelial (trophoblastic) cells, which are 14-60μm in diameter, larger than peripheral blood leukocytes. Enrichment ofcirculating fetal cells followed by genetic diagnostic can be used fornoninvasive prenatal diagnosis of genetic disorders using PCR analysisof a DNA target or fluorescence in situ hybridization (FISH) analysis ofgenes.

Another exemplary application of a microfilter manufactured inaccordance with exemplary aspects of the present invention (e.g. (i)standard microfilters, (ii) nanosurface topography microfilters, (iii)standard microfilters coated with analyte capture elements, and (iv)nanosurface microfilters coated with analyte capture elements) iscollecting or enriching stromal cells, mesenchymal cells, endothelialcells, epithelial cells, stem cells, hematopoietic and non-hematopoieticcells, etc. from a blood sample, collecting tumor or pathogenic cells inurine, and collecting tumor cells in spinal and cerebral fluids. Anotherexemplary application is using the microfilter to collect tumor cells inspinal fluids. Another exemplary application is using the microfilter tocapture analytes bound to latex beads or antigen caused particleagglutination whereby the analyte/latex bead or agglutinated clustersare captured on the membrane surface.

Another exemplary application of a microfilter formed in accordance withexemplary aspects of the present invention (e.g. (i) standardmicrofilters, (ii) nanosurface topography microfilters, (iii) standardmicrofilters coated with analyte capture elements, and (iv) nanosurfacemicrofilters coated with analyte capture elements) is for erythrocytedeformability testing. Red blood cells are highly flexible cells thatwill readily change their shape to pass through pores. In some diseases,such as sickle cell anemia, diabetes, sepsis, and some cardiovascularconditions, the cells become rigid and can no longer pass through smallpores. Healthy red cells are typically 7.5 μm and will easily passthrough a 3 μm pore membrane, whereas a cell with one of these diseasestates will not. In the deformability test, a microfilter having 5 μmapertures is used as a screening barrier. A blood sample is applied andthe membrane is placed under a constant vacuum. The filtration rate ofthe cells is then measured, and a decreased rate of filtration suggestsdecreased deformability.

Another exemplary application of a microfilter formed in accordance withexemplary aspects of the present invention (e.g. (i) standardmicrofilters, (ii) nanosurface topography microfilters, (iii) standardmicrofilters coated with analyte capture elements, and (iv) nanosurfacemicrofilters coated with analyte capture elements) is leukocyte/Redblood cell separation. Blood cell populations enriched for leukocytes(white blood cells) are often desired for use in research or therapy.Typical sources of leukocytes include whole peripheral blood,leukopheresis or apheresis product, or other less common sources, suchas umbilical cord blood. Red blood cells in blood can be lysed. Then theblood is caused to flow through the microfilter with small pores to keepthe leukocytes. Another exemplary application is using the microfilterfor chemotaxis applications. Membranes are used in the study of whiteblood cell reactions to toxins, to determine the natural immunity inwhole blood. Since immunity is transferable, this assay is used in thedevelopment of vaccines and drugs on white blood cells. Anotherexemplary application is using the microfilter for blood filtrationand/or blood transfusion. In such applications, microfilters can be usedto remove large emboli, platelet aggregates, and other debris.

What is claimed is:
 1. A microfilter comprising: a first polymer layerformed from an epoxy-based photo-definable dry film, wherein the firstpolymer layer has flexibility to be disposed on a roll and unrolled; aplurality of first apertures each extending through the first polymerlayer formed by exposing the first polymer layer to a UV light via anoptical mask to obtain a selected shape of said apertures based on saidoptical mask, said first polymer layer forming at least a portion ofsaid microfilter; and a surface on said first polymer layer, whereinsaid surface is modified to include a rough nanosurface, and modified tobe hydrophilic with bonding sites on said surface for reagents.
 2. Themicrofilter of claim 1 further comprising a second polymer layer formedfrom photo-definable dry film and having second apertures extendingthrough the second polymer layer, wherein the second polymer layer hasflexibility to be disposed on a roll and unrolled, and at least one ofthe first apertures and at least one of the second apertures define atleast a portion of a passage extending through the first and secondlayers.
 3. The microfilter of claim 1, wherein the surface is modifiedby changing of a surface energy, the polymer layer having the surfaceenergy raised.
 4. The microfilter of claim 1, wherein said first polymerlayer has a uniform thickness of 5 to 100 microns.
 5. The microfilter ofclaim 1, wherein said first polymer layer has a uniform thickness of 10μm.
 6. The microfilter of claim 1, wherein said selected shape of atleast one of said apertures is one of round, oval, or rectangle.
 7. Themicrofilter of claim 1, wherein said selected shape of said apertures isround with a diameter of 5-20 μm.
 8. The microfilter of claim 1, whereinsaid surface is modified to include said rough nanosurface, and saidrough nanosurface comprises said first polymer layer modified by anoperation selected from the group consisting of changing of the surfaceenergy, altering of the surface topography, and altering of the surfacechemistry, and combinations thereof.
 9. The microfilter of claim 1,wherein said epoxy-based photo-definable dry film is an epoxy-basednegative photo-definable dry film.